This proposal focuses on an in-depth analysis of the structure, function and dynamics of the Ste14p isoprenylcysteine carboxyl methyltransferase (Icmt) from S. cerevisiae, the integral membrane enzyme that is responsible for the posttranslational ?-carboxyl methyl esterification of a large number of cellular proteins that terminate in a C-terminal CaaX motif. CaaX proteins undergo three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the -aaX residues, and methylation of the isoprenylated cysteine by Icmt. Methylation is critical for the proper localization of the CaaX protein K-Ras and may be essential for oncogenic transformation; thus, Icmt may prove to be an excellent chemotherapeutic target. To date, few molecular details are known about the mechanism of Ste14p except that it must accommodate chemically diverse methyl donor and acceptor molecules: the hydrophilic co-factor S- adenosylmethionine (SAM) and a lipophilic isoprenylated protein substrate, respectively. Our long term goal is to understand in mechanistic detail how such an enzyme mediates catalysis at the membrane/cytosol interface. Specifically, our goals in the current proposal are to understand the structure-function relationships of co-factor and substrate recognition by the Icmt enzyme and to elucidate the structural dynamics of the co- factor binding/catalytic loop of the enzyme. As mammalian Icmts have proven intractable to functional purification, we will use Ste14p, as our model enzyme. We have overexpressed, purified and functionally reconstituted milligram quantities of both wild-type and a cysteine-less variant of Ste14p in yeast. We have also generated and characterized a large library of site- directed mutants in conserved residues of His-Ste14p. Using these tools, we are now poised to identify key residues that comprise the binding sites in Ste14p for isoprenylated substrates. We are also set up to identify important residues responsible for binding of the co-factor SAM and to reveal conformational changes that occur in the enzyme upon co-factor and/or lipidated substrate binding by SDSL EPR, guided by a structural homology model of the C-terminal catalytic half of Ste14p that is based on the crystal structure of a bacterial Icmt ortholog. Together, these studies will provide key insights into the nature of substrate and co-factor recognition by Ste14p that will give a better understanding of its mechanism of action. Given the structural, functional and sequence similarities between Ste14p and other Icmts, study of the yeast enzyme should offer general mechanistic insight into the function of other members of this class of enzymes which ultimately may provide the basis for the rational design of anti-cancer drugs targeting human Icmt.